Summary of Work: Calcium signalling in non-excitable cells generally involves mobilization of Ca2+ from intracellular stores by the second messenger, inositol 1,4,5-trisphosphate (IP3). However, although it is clear that the locus of this intracellular store of Ca2+ is either a part of, or is derived from endoplasmic reticulum, the precise locus of these calcium stores and their relationship to other cellular structures is not clear. In addition, the interaction of IP3 with its receptor on these Ca2+ stores often results in complex kinetics of Ca2+ release and reaccumulation, resulting in the appearance of regenerative cytoplasmic intracellular Ca2+ ([Ca2+]i) oscillations. These stores of Ca2+ are important because they are the initial source for the [Ca2+]i signals generated in many different cell types by the actions of hormones, neurotransmitters and growth factors. Changes in the concentration in these stores also initiates a novel signalling pathway that controls such processes as apoptosis, gene expression, and capacitative calcium entry. The focus of research in this project is to identify and localize the intracellular Ca2+ pools involved in cellular signalling, and to understand better the basis for intracellular [Ca2+]i oscillations. We also intend to develop a technique for real-time tracking of the [Ca2+]i in the lumen of intracellular stores. This work will involve a combinationof CCD camera imaging of cells using cytoplasmic and compartmentalized fluorescent calcium dyes together with computer assisted deconvolution, as well as confocal microscopy, and subcellular fractionation.